SNALP Structure Reconsidered

SNALP delivery technology has not only been the subject of a heated fight over ownership and control, but also continued mechanistic, chemistry, and structural investigations.  

In terms of structure, I used to think of SNALP as simple, unilamellar liposomes with RNAi triggers captured in their aqueous interiors.  A patent application by Tekmira (WO2012/000104A1) and a recent paper by the Cullis/AlCana group (Leung et al., 2012) convincingly challenge this view.  Accordingly, SNALP particles are highly electron-dense entities in which pockets of RNAi triggers surrounded by positively charged lipid micelles lipids fill a lipid-enclosed vesicle.  

This new view should guide the future development of the technology, especially the ratio space of its components, targeted delivery approaches, and cytoplasmic release.

Numbers hinting at need for new model

One metric of Tekmira’s results that has always impressed me were its RNAi trigger formulation efficiencies, often well above 90%.  As Leung and colleagues note in their paper, this is at odds with the old simple aqueous encapsulation model which would predict that the likelihoods of an RNAi trigger to be encapsulated or not are roughly equal.  

It now seems that Tekmira’s manufacturing method of rapidly mixing lipids and nucleic acids is at least one critical factor allowing for this remarkable concentration effect.  Other groups largely failed to reproduce such data at least partly because the published method called for the use of costly amounts of reagents.  This is changing, however, due to the use of microfluidic formulation methods as was also practiced by Leung and colleagues. This method likewise allows lipids and nucleic acids to be mixed vigorously and consequently also generates electro-dense structures, but using much smaller volumes.    

Limit-size particles smaller than anticipated

A consequence of the new model is that SNALP LNPs may be as small as 15nm in diameter, whereas previously I subscribed to the view that 40-50nm was the limit.  While a 15nm spherical particles would still have problems in passively getting out of the vasculature in most tissues, it is in a dimension where it might become of interest for additional tissue targets than those that we have assumed to be suitable SNALP targets. 

While it is nice to think that SNALP LNPs has more tissues that it might be able to address, it seems that the more tangible, near-term value of the new insights is in how the particles can be designed to target the existing low-hanging fruit tissues such as liver, sites of inflammation, phagocytes and other cells in the blood/lymph, solid tumors (by systemic delivery), and local applications such as lung epithelia by inhalation.

You could for example imagine that this model makes the post-insertion method of adding a targeting ligand more attractive compared to a co-formulation one (both of which have been considered). Similarly, the model changes the space of lipid and RNAi trigger ratios that should be explored.  It also requires new models for the cytoplasmic release of the RNAi triggers from the endosomes to be considered.

Who was first?

While I do not wish to carry on with pointing out potential tensions between Tekmira, Alnylam, and AlCana, the fact that both Tekmira and the Cullis laboratory (‘AlCana’) came out with essentially the same discovery does not leave me much choice but to briefly comment on the coincidence (note that this clash was set in motion before the settlement).

In the patent application by Tekmira, the priority date is June 30, 2010 2011.  As this is also the international filing date, the invention/discovery must have occurred at least a few months, if not at least a year earlier.  The Leung et al. paper was received by the the Journal of Physical Chemistry approximately 2 1 year later (April 5, 2012).  This suggests that the Tekmira were the first to have these insights.  This, of course, could also be critical for the patent application.  If Tekmira were able to get patent protection for such ‘non-lamellar’ LNPs, this could be an important one and somewhat replace in importance the Semple/Wheeler patent estate which are about to expire over the next years.

The new findings illustrate that SNALP LNP delivery continues to be an area a rapid progress.  One would make a mistake to assume that the MC3 formulations currently in the clinic are as good as it gets and that other somewhat overlapping, and therefore competing approaches such as Arrowhead’s DPCs are about to catch up.  Here’s hoping that with the leading delivery technologies having so much obvious room to mature, 2013 will be at least as exciting if not more so than 2012 for RNAi Therapeutics.

Argonaute Gymnastics May Guide siRNA Design (Journal Club)

Argonautes are at the heart of all RNAi-related small RNA-regulated processes. A structural understanding of how they load, activate, and utilize siRNAs to seek out and repress target mRNAs would therefore prove invaluable for the rational design of potentially ever more potent and specific siRNAs. The high-impact journal Nature appears to agree, as it has been publishing at an astonishing frequency collaborative work from the Patel and Tuschl labs at Rockefeller on beautiful structures of Argonautes at the various stages of their catalytic cycle.

In the latest publication in this series, they catch Argonaute with the activated guide strand bound to first a short (12 nucleotides), and then an elongated target RNA (15 nucleotides and longer). What is remarkable is that the enzyme undergoes a substantial structural rearrangement when binding to the longer target, most notably accompanied by the popping out of the guide strand 3’ end from the PAZ pocket where it usually sits in the single-stranded form. This is thought to reflect a 2-state mode of binding fully complementary target RNAs and may differ in terms of structural re-arrangement to what happens in the setting of microRNA target recognition which is the cause for siRNA off-targeting. What is always neat to see in those structures is how they rationalize a lot of preceding molecular biology and bioinformatics work. In this case, this includes the importance of the ‘seed’ nucleotides 2-8, the sensitivity of RNAi target cleavage to mismatches and modifications, and the relative unimportance of the 3’ terminal guide bases 17-21.

The structure may help to expand on work that shows that it is possible to design small RNAs for which target cleavage and translational repression can be functionally separated. Previous work by Dharmacon (now part of Thermo Fischer) for example showed that methylation at position 2 of the guide strand abrogates its ability to translationally repress targets, while leaving cleavage intact. While those siRNAs should be highly specific, the somewhat slow adoption of this strategy indicates to me that more work needs to be done so that similar strategies are more generalizable. Based on the two-state model and present structure, such modification-induced distortions may critically affect seed-binding energy on which translational repression is so much dependent, while the conformational shift that accompanies the recognition of fully complementary on-targets, may lock the Argonaute-guide-target complex into a stable cleavage-competent conformation. The structure may help define universally applicable modification strategies that are targeted towards the 5’ end of the guide strand.

The work also demonstrates how target cleavage is sensitive to modifications around the cleavage site, and finds certain positions better tolerate them than others. This may prove to be important for aiding siRNA activation which is thought to primarily function via cleavage of the passenger strand. Finally, the series of Argonaute structures with bound guide strand provided some nice snapshots of how the 5’ end is nestled in the so-called MID-domain of the enzyme. It is not far-fetched to believe that by optimizing this interaction (e.g. 5’ end modification, nature of the base itself etc), the capture and longevity of the guide strand in the Argonaute protein can be increased with possibly increased duration of activity of the activated enzyme complex, and in the case of single-stranded RNAi, improved loading, too. Finally, this type of structural work is also highly relevant for research into how to reconcile the need for modifications for increasing siRNA stability and avoiding innate immune responses with RNAi activity.

The next major structural insight that should guide design strategies for therapeutic RNAi will be from the elucidation of mammalian Argonaute structures (humans have 4 Argonautes), as the present work focused on archaebacterial Argonautes due to the relative ease of working with them. These, however, will differ in several important aspects from mammalian Argonautes, for example in that they utilize DNA as guides and maybe even as their natural targets. Nevertheless, with such healthy progress on the molecular understanding of RNAi, the efficiency of the RNAi Therapeutics drug development platform will only increase, and it is fun to imagine what it could look like in 10 years, at which point we may be able to watch a movie on the life of an Argonaute protein by the Patel lab.